The related fungal species Cryptococcus neoformans and Cryptococcus gattii frequently infect humans, causing life-threatening lung and brain infections. Although often considered opportunistic pathogens, these organisms can cause disease in hosts with both compromised and normal immunity. More than 1,000,000 infections occur worldwide annually, leading to >620,000 deaths comprising more than one-third of all HIV/AIDS-related deaths. A specialized genomic region, the mating type locus (MAT), governs cell identity, sexual reproduction, infectious spore production, and virulence. We previously discovered that the Cryptococcus MAT locus is a large, complex gene cluster, and we proposed to elucidate how this unusual suite of sex- and virulence-determining genes evolved from a simpler nonpathogenic ancestral state. Our comparative phylogenomic studies provide evidence that 1) the bipolar/unipolar mating system is a shared characteristic of these two Cryptococcus pathogenic species, and 2) the inbreeding bipolar/unipolar system evolved from an ancestral outbreeding tetrapolar system via gene acquisition and translocation-driven fusion. Similar transitions have occurred in other fungal pathogens of plants and animals, suggesting convergent evolution in concert with host adaptation. These studies illustrate general principles of gene cluster evolution and forces by which recombination has forged the genomes of microbial pathogens. Our studies supported by this award defined the structure, function, and evolution of the MAT locus. In the prior award period, we 1) cloned and sequenced the bipolar MAT locus from pathogenic Cryptococcus species; 2) discovered tetrapolar sexual cycles and MAT loci of closely related nonpathogenic species; and 3) found the two MAT loci of the nonpathogen Cryptococcus amylolentus lie on different chromosomes and are both centromere-linked. We hypothesize the tetrapolar-bipolar transition occurred concomitant with pathogen emergence via two steps. First, genes were recruited into two unlinked MAT loci; second, chromosomal translocation fused the MAT loci. Our recent studies reveal novel MAT features allowing us to propose new aims to test these hypotheses. Aim 1 focuses on MAT locus structure and evolution. We will sequence genomes, define centromeres, and test the hypothesis that inter-centromeric recombination drove fusion of unlinked ancestral MAT loci and key chromosomal translocations that punctuate pathogen evolution. Aim 2 focuses on MAT locus functions. We will address 1) mechanisms governing uniparental mitochondrial inheritance that restrict mitochondrial genome recombination and impact mitochondrial dynamics that promote replication inside macrophages and pathogenesis, and 2) roles of essential diverged ribosomal protein paralogs in development and virulence. Not only do these studies advance our understanding of the dynamic evolution of microbial genomes, but given the association with specific gene clusters and virulence, they also have direct implications for infectious disease treatment and prevention.